Lyme disease is the most frequently reported arthropod-borne disease in the United States and Europe (reviewed in ref (Steere, 2004). Serological assays are the most common laboratory tests used to confirm or support a diagnosis based on clinical features and epidemiologic circumstances (reviewed in (Bunikis, 2002; Aguero-Rosenfeld, 2005). Direct detection of the organism by cultivation, histology of a biopsy, or by an approved and validated polymerase chain reaction assay is generally preferable to serological assays for definitive confirmation of a clinical diagnosis but these procedures are uncommon in practice and not likely become widely used for the foreseeable future.
A clinical diagnosis of Lyme disease based on the observation of a characteristic skin rash and suitable epidemiologic features (e.g. exposure to ticks in an endemic area during the season of transmission) can have high accuracy (Steere, 2004). But in the absence of a skin rash (˜20-30% of cases) diagnosis of early Lyme disease solely based on clinical and epidemiologic features is more difficult. Accurate diagnosis of early infection without the typical skin rash is important, because oral antibiotic treatment at this point is usually successful and will prevent the more serious manifestations of disseminated disease and late disease. Serologic assays for late disseminated Lyme disease are also important to help confirm a clinical diagnosis of potentially-treatable chronic infection. But a commonly used, if not recommended, practice is to use a serologic assay to “rule out” B. burgdorferi infection as an explanation of what may be long-standing symptoms, such as chronic joint pain, headache, cognitive problems, and fatigue. For diagnosis of early infection, a sensitive test is desirable to identify the infection at the earliest and most easily treatable point of the infection. For diagnosis of late disease, high sensitivity is also desirable but improved specificity is especially important because the test in practice is often applied in circumstances in which the a priori likelihood of B. burgdorferi infection is low (Bunikis, 2002).
Currently available commercial assays in the United States are either based on whole bacteria cell extracts, such as the enzyme-linked immunoabsorbent (ELISA) and Western blot assays, or on a single antigen ELISA such as the C6 peptide of the VlsE protein (Aguero-Rosenfeld, 2005). The whole cell assays are usually used as a 2-tiered test. First, a more sensitive, typically a whole cell ELISA, is used. This is followed by the more specific Western blot, if the ELISA is positive or equivocal (Control, 1997). Together these assays have served for years as the standard for serodiagnosis, but there remain trade-offs between sensitivity and specificity to minimize false-positive results. One drawback of the 2-tiered, sequential test procedure is the time it takes and the greater expense for two assays. Another problem with whole cell assays is a lack of standardization between tests of different manufacturers. The variables include different strains of B. burgdorferi that are used, different conditions for cultivating the organisms, and different methods for identifying the key antigens on blots.
Assays based on single proteins, such as the flagellin protein FlaB, or combinations of recombinant proteins are available in Europe (Hansen, 1988; Kaiser, 1999; Heikkila, 2003). In general, these have shown sensitivities and specificities approximately equivalent to the 2-tiered procedure. The recombinant antigens used singly or in combination are those that had been previously identified in whole cell Western blot assays using in-vitro cultivated cells. In the United States the most common subunit assays use a single peptide (called C6) of the VlsE protein or the full-length recombinant VlsE protein (Bacon, 2003). In some test formulations these single antigen assays had sensitivity for different stages of infection that was as good as the 2-tier procedure and better specificity (Lawrenz, 1999; Liang, 1999). But in other, more recent studies, including some from Europe, either the specificity or sensitivity of single antigen assays was not as good as tests based on two or more antigens or a 2-tiered procedure (Peltomaa, 2004; Marangoni, 2005; Goettner, 2005).
Perhaps the most important problem with currently available whole cell-based assays is that they utilize for their substrates bacteria that have been grown in vitro. The accumulated evidence=unequivocally shows that cells grown in vitro differ with respect to the expression of several proteins from cells recovered from infected animals (Fikrig, 1997; Gilmore, 2001; Salazar, 2005). While certain in vivo conditions can be duplicated to some extent in vitro by altering growth conditions, such as pH or cell density, there remain many proteins that appear to be only expressed in an infected animal or untreated patient.